Physico-chemical and antigenic properties of tetanus and diphtheria toxoids and steps towards improved stability

Physico-chemical, antigenic and immunogenic properties may be altered during microencapsulation of antigens and their release from poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres. Here, the physico-chemical, conformational and antigenic stability of tetanus and diphtheria toxoids was studied in aqueous solutions stressed by elevated temperature and the presence of lactic and glycolic acids. Further, the stabilising effect of albumin was investigated. The analytical tools used were fluorimetry, circular dichroism spectroscopy, turbidimetry, electrophoresis and ELISA. Elevated temperatures altered the physico-chemical and antigenic properties of the toxoids to a greater extent than the acids (50 mM) did. Substantial unfolding and chemical changes of tryptophan were observed upon 1-4 weeks of incubation at 60 degreesC. At 4 degreesC, only minor conformational changes were observed, even in the presence of the acids. Furthermore, 40% of the tetanus toxoid antigenicity was lost after 7 days at 37 degreesC. This loss increased in the presence of the acids. At 60 degreesC, the antigenicity had completely vanished. Very importantly, 0.5% albumin preserved the tetanus antigenicity over 6 weeks' incubation at 37 degreesC, regardless of the presence of glycolic acid. This qualifies albumin as potential stabilising additive for toxoid loaded poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres.